In collaboration with clinical investigators in NIDDK, we are continuing the antiviral immune response of patients who have accidentally been exposed to HCV. To date, more than 70 subjects have been enrolled in the study. All enrolled subjects are followed for virological markers, serum antibodies and cellular immune responses to HCV on the day of exposure and at weeks 2, 4, 6, 12, 24 and 48 after exposure. No subject tested positive for HCV-RNA (Amplicor Assay, Roche Diagnostics) or HCV antibodies (2nd generation EIA, Abbott Laborat.) at any time point during the study. Peripheral blood mononuclear cells are being tested for HCV-specific proliferation using recombinant HCV core, NS3, NS4, NS5A and NS5B proteins. Direct, ex vivo effector functions of HCV-specific CD4+ and CD8+ T cells are assessed by IFN-gamma ELISpot analysis using 228 overlapping 15-mer peptides spanning the HCV core, NS3, NS4A and NS4B proteins. At this time, about 70% of the immunological assays have been completed showing an increase of HCV-specific proliferative T cell responses against structural and nonstructural HCV proteins in a subgroup of subjects after exposure. This HCV-specific proliferative response was followed by HCV-specific IFN-gamma production, thus indicating direct, ex vivo effector functions of the responding cells. HCV-specific T cell responses returned to baseline levels in most subjects within 12 weeks after exposure. These preliminary results demonstrate that low level exposure to HCV can induce and/or boost HCV-specific T cell responses in the absence of antibody production and detectable viremia. Thus, cellular immune responses might be a more sensitive indicator of HCV exposure than humoral responses. The data also suggest that repeated exposure to HCV is sufficient to immunize the exposed individuals without causing clinically evident infection. [unreadable] [unreadable] In collaboration with intra- and extramural investigators, we are also analysing cellular and humoral immune responses prospectively in individuals who have developed acute and chronic HCV infection. The aim of these studies is to compare responses in persons whose infection resolves with those who progress to chronic infection. An analysis of T cell reactivity of patient H, the source of the prototype HCV H77 strain, who became infected with HCV through blood transfusion in 1977 and developed chronic infection, revealed strong reactivity against four epitopes, two of them located in a single peptide. All of these epitopes coincide with residues that show phylogenetically informative changes over time, which completely abrogated T cell reactivity in 3 of 4 epitopes demonstrating that the HCV quasispecies population in the setting of chronic HCV infection escapes from recognition by CD4 and CD8 T cells. Whereas escape from cellular immune responses had previously only been shown for CD8 T cells, we here demonstrate two instances of viral escape in a CD4 epitope. These data strongly suggest that CD4 and CD8 T cell in addition to B cell-mediated selective pressures impact on HCV glycoprotein evolution.[unreadable] [unreadable] Finally, an ongoing study examines whether residual HCV RNA may still be detected in the plasma of patients who are convalescent from hepatitis C as evidenced by conventional laboratory assays and liver function tests. Using highly sensitive reverse transcription-polymerase chain reaction with a sensitivity level of <40 copies/ml to analyze plasma samples from 67 sustained treatment responders and 25 spontaneoulsy recovered patients, we detected residual HCV RNA in convalescent-phase plasma of 11/67 (15%) sustained treatment responders as compared to 0/25 spontaneoulsy recovered patients. Sequence analysis demonstrated presence of the same genotype as prior to treatment. Genotype 1b was identified in four cases, and genotypes 1a and 2b were identified in two individuals each, whereas genotype 3a was prominent in one case. These results imply that HCV RNA can persist at very low levels in the plasma for many years after apparent complete treatment-induced resolution of hepatitis C. However, it is unclear at present whether these low levels of HCV RNA pose risks for transmission of the virus or for the reactivation of infection.